Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Catalysis

Background:

• Small ribozymes, like Oryza sativa twister, undergo spontaneous RNA self-cleavage upon achieving their active conformation.
• Studying the precise timing of ribozyme folding and self-cleavage is challenging due to the rapid conversion of the active ribozyme to its product.

Purpose of the Study:

• To develop a method for precisely controlling the timing of ribozyme folding and self-cleavage.
• To resolve the dynamic interplay between ribozyme folding and RNA self-cleavage events at the single-molecule level.

Main Methods:

• Synthesis of a photocaged nucleotide releasing guanosine upon blue light exposure within microseconds.
• Application of the photocaged nucleotide to O. sativa twister ribozymes.
• Combined use of the light-activated tool with single-molecule fluorescence detection for real-time observation.

Main Results:

• Achieved spatial (75 µm) and temporal (≤30 ms) control over ribozyme folding and self-cleavage.
• Observed that the folded, pre-cleaved state is unstable and can unfold if the RNA does not react.
• Determined that Mg²⁺ and Mn²⁺ enhance ribozyme efficiency by promoting transitions to the active conformation, not by stabilizing ground or product states.

Conclusions:

• The developed light-controlled system enables precise temporal and spatial control over single RNA molecule folding.
• This tool provides unprecedented insights into the dynamics of ribozyme folding and catalytic activity.
• The methodology is applicable to controlling and studying other nucleic acid systems with high temporal resolution.